Crone, S. A. Project Summary The proposed studies investigate the role of spinal V2a neurons in the control of auxiliary (non- diaphragmatic) respiratory muscles (ARMs) for inspiration. These muscles are normally used to increase ventilation during exercise, but they are also used to augment diaphragm function after injury or disease. Despite the importance of ARMs for enhancing ventilation or compensating for loss of diaphragm function, little is known about the neural circuits that drive their activity in either health or disease. ARM activity increases ventilation in ALS model mice at early disease stages, but there is a central deficit that prevents activation of ARMs for breathing at late disease stages, despite the fact that these muscles are active and functional for voluntary behaviors. Either increasing (through Gq signaling pathways) or decreasing (through Gi signaling pathways) the excitability of V2a neurons is able to increase ARM activity at rest in healthy and ALS model mice. Thus, these neurons are a potential target to increase ARM activity and ventilation in patients with ALS, other neuromuscular diseases, or spinal cord injury. However, the V2a class of neurons appears to be composed of subtypes that play distinct roles in the control of breathing. In order to develop therapies to improve breathing by altering the activity, preventing degeneration, or replacing degenerated V2a neurons, it is necessary to determine the location and molecular subtypes of V2a neurons that impact ventilation and to understand their specific roles. Aim 1 will use transgenic and viral strategies to increase excitability, decrease excitability, or ablate V2a neurons in cervical, thoracic, or lumbar spinal cord to identify how respiratory muscle activity is regulated by V2a neurons at different segmental levels. Aim 2 will investigate the molecular diversity of V2a neurons within the cervical cord by investigating differences in connectivity and function within respiratory circuits of a medial V2a subtype and a lateral V2a subtype. Aim 2 will also asses the effects on ventilation of altering excitability of only V2a neurons with direct excitatory connections to respiratory motor neurons. Aim 3 will assess the long- term impact that degeneration of V2a neurons has on breathing by prematurely ablating cervical V2a neurons in a mouse model of ALS. The potential benefit (and hazards) of chronically increasing the excitability of V2a neurons on ARM activity, motor neuron degeneration, and ventilatory health in ALS model mice will also be assessed. By accomplishing these aims, we will pinpoint the location, molecular identity, and connectivity of spinal V2a neurons that pattern respiratory muscle activity as well as assess the potential of pharmacologically altering the excitability of V2a neurons to improve breathing in a mouse model of ALS.